Upon large-scale expression of therapeutic proteins, according to characteristics of host cells or target proteins, a target protein may vary in expression level, water solubility, expression sites, modification, and the like. Thus, the most suitable expression system for a target protein must be selected to establish an effective production system. Glycoproteins currently constitute about 70% of the recombinant therapeutic protein market, playing a leading role in the market. The components and structure of N-linked sugar moieties, which are attached to asparagine residues of glycoproteins, have been found to be major factors in determining the efficacy and stability of glycoproteins (Koury, M., Trends Biotechnol. 21, 462-464 (2003)). Animal cell culture technologies, which are capable of producing glycoproteins containing sugar moieties most similar to human's, are currently leading the market. However, there are several drawbacks to animal cell culture systems, which include low yield, high cost due to expensive culture media, risk of infection with viruses and prions, and a long period of time required to establish stable cell lines. Thus, animal cell culture systems have limited application in recombinant glycoprotein production.
As an alternative to animal cell culture systems, yeast expression systems have some advantages of being cost-effective, rapidly growing to high cell density in chemically defined medium, being easily genetically engineered, producing high yield of recombinant proteins, having no risk of infection with human or animal pathogens, and ensuring easy protein recovery. Moreover, as lower eukaryotes, yeasts share the early stages of the N-linked oligosaccharide of higher animal cells, and so could be utilized to produce several glycoproteins with therapeutic purpose. However, glycoproteins produced from yeast expression systems contain nonhuman N-glycans of the high mannose type, which are immunogenic in humans and thus of limited therapeutic value. In particular, this yeast-specific outer chain glycosylation of the high mannose type, denoted hyperglycosylation, generates heterogeneous recombinant protein products, which may make the protein purification complicated or difficult. Further, the specific activity of hyperglycosylated enzymes may be lowered due to the increased carbohydrate level (Bekkers et al., Biochem. Biophy. Acta. 1089, 345-351 (1991)).
To solve the above problems, there is a need for glycoengineering, by which the yeast glycosylation pathway is remodeled to express glycoproteins having glycan structure similar to that of human glycoproteins. Glycoengineering was first applied to the traditional yeast, Saccharomyces cerevisiae which has the heavily hypermannosylated N-glycan structure composed of additional to 200 mannose residues attached to the core oligosaccharide and decorated with the terminal α-1,3-linked mannoses highly immunogenic when injected to human body. Compared to S. cerevisiae, the methylotrophic yeasts, Hansenula polymorpha and Pichia pastoris, are shown to produce N-linked glycans with shorter mannose outer chains and no α-1,3-linked terminal mannose (Kim et al., Glycobiol. 14, 243-251 (2004)). Therefore, the methylotrophic yeasts are considered superior expression hosts to the traditional yeast, S. cerevisiae, for the production of glycoproteins with therapeutic value. In addition, their excellent capacity in secreting recombinant proteins into the medium makes these methylotrophic yeasts favorable host systems for secretory protein production in the economical perspects.
H. polymorpha is a well known host for the production of recombinant hepatitis B vaccine, which has been approved for therapeutic use and already available on the market. At present, other H. polymorpha-derived therapeutic recombinant proteins, such hirudin, elafin, and insulin, are launched in the market, demonstrating high potential of H. polymorpha as a practical host for the production of therapeutic recombinant proteins (Kang and Gellissen, Production of Recombinant proteins. Ed. G. Gellissen, pp. 111-136 (2005)) However, technologies involving the remodeling of the yeast glycosylation pathway for the production of glycoproteins having human-type glycans have been mainly developed in S. cerevisiae, which is a well-characterized yeast, and P. pastoris, based on which a protein expression system is available (WO0114522, WO0200879, WO04003194, US2005/0170452, Wildt and Gerngross, Nature Rev. Microbiol. 3, 119-128 (2005)). In contrast, studies employing H. polymorpha in glycoengineering have seldom been conducted.
As an example of studies employing H. polymorpha in glycoengineering, the present inventors, prior to the present invention, cloned HpOCH1 and HpOCH2 genes, which play critical roles in the outer chain synthesis of H. polymorpha, and developed a process for producing a recombinant glycoprotein having a non-hyperglycosylated glycan structure using mutant strains having a disruption in any one of the genes (Korean Pat. Application No. 2002-37717 and No. 2004-6352, PCT Application PCT/KR2004/001819). However, a trimannose core structure containing three mannoses and two N-acetylglucosamine(Man3GlcNAc2), which is the minimal common backbone of N-glycans, should be made in order to express glycoproteins having human compatible hybrid- and complex-type glycans.
In this regard, the present inventors identified a novel gene (HpALG3) coding for dolichyl-phosphate-mannose dependent α-1,3-mannosyltransferase, which is a key enzyme involved in the early stages of lipid-linked oligosaccharide biosynthesis prior to oligosaccharide addition to a glycoprotein, from the methylotrophic yeast H. polymorpha, and found that the manipulation of the gene alone or in combination of one or more genes, each coding for an enzyme involved in glycosylation, enables various manipulation of the glycosylation process of H. polymorpha and the preparation of glycoproteins having human-type glycans, thus leading to the present invention.